Bacterial Cell Staining and Preparation for Immunological Studies

First, harvest the bacterial cultures at an exponential phase by centrifugation at 13,000 times g for two minutes at room temperature. After washing the cell pellet with one milliliter PBS, resuspend the bacterial pellet in one milliliter PBS.

Determine the concentrations of bacterial suspension by measuring optical density at 600 nanometers. Then, for staining the bacterial suspension, add two microliters of the 500-fold concentrated stock green or red staining dye into one milliliter of bacterial suspension to dilute the dye one-fold. Incubate the cells at room temperature for 30 minutes with gentle rotation in the dark.

After 30 minutes, centrifuge the stained bacteria at 13,000 times g for two minutes, and resuspend the pellet in one milliliter of PBS. Collect the stained bacterial cells by centrifugation at 13,000 times g for two minutes. Resuspend the pellet in one milliliter of fresh F-12K medium, and measure the optical density of each culture at 600 nanometers. Subsequentially dilute the cultures to the desired concentrations, based on the multiplicity of infection and host cell concentration.